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Directed differentiation of embryonic origin–specific vascular smooth muscle subtypes from human pluripotent stem cells

Nature Protocols volume 9, pages 929–938 (2014)Cite this article

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Abstract

Vascular smooth muscle cells (SMCs) arise from diverse developmental origins. Regional distribution of vascular diseases may, in part, be attributed to this inherent heterogeneity in SMC lineage. Therefore, systems for generating human SMC subtypes of distinct embryonic origins would represent useful platforms for studying the influence of SMC lineage on the spatial specificity of vascular disease. Here we describe how human pluripotent stem cells can be differentiated into distinct populations of SMC subtypes under chemically defined conditions. The initial stage (days 0–5 or 0–7) begins with the induction of three intermediate lineages: neuroectoderm, lateral plate mesoderm and paraxial mesoderm. Subsequently, these precursor lineages are differentiated into contractile SMCs (days 5–19+). At key stages, the emergence of lineage-specific markers confirms recapitulation of embryonic developmental pathways and generation of functionally distinct SMC subtypes. The ability to derive an unlimited supply of human SMCs will accelerate applications in regenerative medicine and disease modeling.

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Figure 1: Timeline outlining the generation of SMC subtypes (NE-SMC, LM-SMC and PM-SMC) from hPSCs.
Figure 2: Phase-contrast images of hPSCs during the course of differentiating into SMC subtypes.
Figure 3: Flow cytometry analysis of lineage-specific markers on the precursor populations.
Figure 4: Immunocytochemistry of SMC markers on precursor lineages and SMC subtypes.

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Acknowledgements

We thank L. Vallier for the established work on neuroectoderm differentiation. S.S. is funded by the British Heart Foundation (FS/13/29/30024 and NH/11/1/28922) and the Cambridge National Institute for Health Research Cambridge Biomedical Research Centre. C.C. is supported by an Independent Fellowship from the Institute of Molecular and Cell Biology (Singapore). A.S.B. is supported by the British Heart Foundation (FS/12/37/29516).

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Authors and Affiliations

  1. Institute of Molecular and Cell Biology, Agency for Science, Technology and Research, Singapore.,

    Christine Cheung

  2. The Anne McLaren Laboratory for Regenerative Medicine and Wellcome Trust—Medical Research Council Cambridge Stem Cell Institute, University of Cambridge, Cambridge, UK

    Andreia S Bernardo, Roger A Pedersen & Sanjay Sinha

  3. Division of Cardiovascular Medicine, University of Cambridge, Addenbrooke's Hospital, Cambridge, UK

    Sanjay Sinha

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  1. Christine Cheung
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Contributions

C.C. and S.S. designed the protocol. C.C. performed the differentiation experiments, analyzed the data and wrote and prepared the manuscript. A.S.B. and R.A.P. established the mesoderm induction conditions and coedited the manuscript. S.S. supervised the project and edited the manuscript.

Corresponding authors

Correspondence to Christine Cheung or Sanjay Sinha.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Methods

Immunofluorescence and flow cytometry (PDF 112 kb)

Supplementary Table 1

Approximate yields of SMC subtypes from hPSCs. Values represent (mean ± s.d.). (PDF 113 kb)

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Cheung, C., Bernardo, A., Pedersen, R. et al. Directed differentiation of embryonic origin–specific vascular smooth muscle subtypes from human pluripotent stem cells. Nat Protoc 9, 929–938 (2014). https://doi.org/10.1038/nprot.2014.059

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